Our goal is to understand how the nanoscale arrangement of kinetochore proteins shapes its functional and regulatory mechanisms. The kinetochore is a macromolecular motor that drives chromosome movement and ensures their accurate segregation during cell division. Kinetochore force generation required for chromosome movement is critical for inheritance of a complete genome by both daughter cells. Kinetochore misregulation leads to chromosomal instability, which has been linked to tumorigenesis, developmental defects, as well as age- related infertility. Therefore, definition of the biophysical mechanism of kinetochore force generation is necessary to develop a mechanistic understanding of disease relevant mutations in kinetochore proteins. Although the last decade has witnessed tremendous progress in our understanding the protein composition of the kinetochore, a mechanistic understanding of its function as a force generator remains elusive. The primary obstacle in further progress is a lack of understanding of the molecular architecture of the kinetochore. Therefore, we propose a novel 'architecture-function' approach to establish mechanistic link between kinetochore architecture and its function. Aim 1: Develop a new fluorescence microscopy method to reconstruct the nanoscale kinetochore architecture. We have developed a new technique to determine nanoscale distribution of proteins in live cells. Our preliminary reconstruction of kinetochore architecture suggests an integrative model of how the kinetochore generates microtubule polymerization and depolymerization coupled force. Our technique will be useful for determining the architecture of other cellular machines. Aim 2: Determine how the location of force generating molecules defines their function. We will subject our new model to an 'architecture-function' analysis, wherein we will study the impact of changes in kinetochore architecture on its function. This work will define the biophysical principles of force generation by the kinetochore. Aim 3: Define the minimal architectural specification for the kinetochore. We will use in vitro experiments and artificial kinetochore protein assemblies to determine the necessary and sufficient architectural features of a key kinetochore protein, Ndc80, for reconstituting its distribution and function observed in vivo. This work will establish a framework for building artificial kinetochores in cells.